Rho-family of GTPases, such as Rac, are central coordinators of signaling pathways that regulate actin cytoskeleton, cell survival and cell motility. Integrins, which are receptors for extracellular matrix proteins, regulate Rac in two ways. First, integrins control the GTP loading of Rac. Second, integrins regulate membrane targeting of GTP-loaded, activated Rac. Both of these events are essential for Rac to activate downstream effectors. Our preliminary data demonstrate that the adapter protein Crk and its binding partner DOCK180 are crucial for both of the regulatory steps in the activation of Rac signaling. In the present application, we will elucidate the mechanisms of the Crk/DOCK180 complex leading to Rac activation. In Aim 1, we will investigate the mechanism by which integrins control the membrane targeting of active Rac. Our preliminary studies indicate that Crk and Rac become recruited to lipid rafts upon integrin ligation and that this recruitment is essential for activation of Rac signaling. We will investigate the mechanisms of Crk/Rac localization to lipid rafts and characterize the events through which cell adhesion regulates lipid rafts. It is anticipated that these studies will reveal novel aspects of not only integrin signaling, but also of lipid raft regulation and function. In Aim 2, we will study the molecular mechanism by which GTP loading and activation of Rac is controlled. Our preliminary data demonstrate that DOCK180 functions as a novel guanine nucleotide exchange factor (GEF) for Rac. In this aim, a multidisciplinary approach ranging from cellular to structural biology will be undertaken to elucidate the regulation of DOCK180 activity in detail. At present, nothing is known about the function of mammalian DOCK180 in vivo. Our goal in Aim 3 is to determine the physiological role of DOCK180 by generating a DOCK180-deficient mouse model. Subsequent studies in mouse cancer models will address the significance of DOCK180 in tumor initiation and progression in vivo. This work combined with studies proposed in the other two aims will enable us to fully understand the mechanisms and significance of the Crk/DOCK180 complex in regulating Rac signaling. These studies will contribute to our fundamental understanding of how integrins regulate Rho GTPases. These regulatory events are central to cell migration, survival and growth in normal human physiology and diseases, such as cancer.